Gut microbiota dysbiosis-induced activation of the intrarenal renin-angiotensin system is involved in kidney injuries in rat diabetic nephropathy

Abstract

Some studies have shown that gut microbiota along with its metabolites is closely associated with diabetic mellitus (DM). In this study we explored the relationship between gut microbiota and kidney injuries of early diabetic nephropathy (DN) and its underlying mechanisms. Male SD rats were intraperitoneally injected with streptozotocin to induce DM. DM rats were orally administered compound broad-spectrum antibiotics for 8 weeks. After the rats were sacrificed, their blood, urine, feces, and renal tissues were harvested for analyses. We found that compared with the control rats, DM rats had abnormal intestinal microflora, increased plasma acetate levels, increased proteinuria, thickened glomerular basement membrane, and podocyte foot process effacement in the kidneys. Furthermore, the protein levels of angiotensin II, angiotensin-converting enzyme, and angiotensin II type 1 receptor in the kidneys of DM rats were significantly increased. Administration of broad-spectrum antibiotics in DM rats not only completely killed most intestinal microflora, but also significantly lowered the plasma acetate levels, inhibited intrarenal RAS activation, and attenuated kidney damage. Finally, we showed that plasma acetate levels were positively correlated with intrarenal angiotensin II protein expression (r = 0.969, P < 0.001). In conclusion, excessive acetate produced by disturbed gut microbiota might be involved in the kidney injuries of early DN through activating intrarenal RAS.

Keywords: broad-spectrum antibiotics; diabetic nephropathy; gut microbiota; plasma acetate; renin–angiotensin system.

Fig. 1

General parameters of the three groups. a Blood-glucose levels. b Plasma insulin levels. *P < 0.01 compared with the control group, ^#P < 0.05 compared with the DM group.

Fig. 2

The changes in gut microbiota in the three groups. a Taxonomic cladogram derived from LEfSe analysis of 16S sequences. Red shaded areas indicate control-enriched taxa, green shaded areas indicate DM-enriched taxa, and blue shaded areas indicate DM + AB-enriched taxa. b–e Subgroup sequencing analysis of gut microbiota. Class A indicates the control group, Class B indicates the DM group, and Class C indicates the DM + AB group. The solid and dashed lines indicate the average and median relative abundance, respectively. b The relative abundance of Blautia in the gut microbiota of the three groups. c The relative abundance of Roseburia in the gut microbiota of the three groups. d The relative abundance of Paraprevotella in the gut microbiota of the three groups. e The relative abundance of Bacteroides in the gut microbiota of the three groups.

Fig. 3

Treatment with antibiotics significantly reduced the level of plasma acetate. Plasma level of acetate. *P < 0.01 compared with the control group, ^#P < 0.05 compared with the DM group.

Fig. 4

The effect of gut microbiota on renal indicators of incipient DN. a Kidney weight-to-body weight ratios. b 24-h urine protein. c Blood urea nitrogen (BUN) level. d Blood creatinine. *P < 0.01 compared with the control group, ^#P < 0.05 compared with the DM group.

Fig. 5

The effect of gut microbiota on renal injuries of incipient DN. a Pathological changes were assessed by PAS staining (original magnification, ×400). b The score of mesangial expansion was determined from histology sections. ***P < 0.01 compared with the control, ^#P < 0.05 compared with the DM group. c Changes in podocytes were evaluated by electron microscopy (original magnification, ×12 000). d The changes in glomerular endothelium glycocalyx were assessed by WGA staining (original magnification, ×400). e WT-1 and nephrin protein expression was evaluated by immunofluorescent staining (original magnification, ×400). The arrows indicate glomerular endothelium glycocalyx. f Quantitation of immunofluorescence staining for WT-1. *P < 0.05; compared with the control; ^##P < 0.01 compared with DM. g Quantitation of immunofluorescence staining for nephrin. ***P < 0.001; compared with the control; ^#P < 0.05 compared with DM.

Fig. 6

Intrarenal RAS is activated in early DN. Plasma renin activity level (a), Ang I level (b), and Ang II level (c). The protein expression levels of RAS were measured by Western blotting (d). The histograms represent the mean ± SD of the densitometric scans of the protein bands normalized to β-actin (e). Correlation analysis of plasma acetate levels and intrarenal Ang II expression levels (f) (r = 0.969, P < 0.001). *P < 0.01 compared with the control group, ^#P < 0.05 compared with the DM group.